In previous studies on the mechanism of vitamin D-dependent Ca2+ transport across the intestine, we prepared membrane vesicles from rat intestinal epithelial cells (enterocytes) and observed that Golgi membrane vesicles demonstrated the highest Ca2+ uptake when compared with lateral-basal (L-B) and microvillus (MV) membrane vesicles. This Ca2+ uptake was specifically dependent on 1,25(OH)2D3, the active metabolite of vitamin D. Recent studies, supported by this grant, have shown that this Ca2+ uptake was due to binding to non-esterified fatty acids (NEFAs) in the membranes and was associated with an increased phospholipase A activity. Membranes prepared from isolated enterocytes had markedly decreased Ca2+ binding and better demonstrated a vitamin D-dependent ATP-requiring Ca2+ uptake, particularly for duodenal villus cells (as contrasted with crypt cells). To pursue these findings further we propose: 1) to define the role of NEFAs, phospholipids and phospholipases in the molecular mechanisms of sequestering calcium during its absorption by the intestine, 2) to purify the Ca2+-ATPase of L-B membrane presumed responsible for ATP-dependent Ca2+ uptake, and 3) to study the molecular mechanisms by which 1,25(OH)2D3 effects Ca2+-ATPase function by either a genomic mechanism or through a post-translational activation of the enzyme. We will also try to determine if 1,25-(OH)2D3 stimulates the synthesis or appearance of proteins in Golgi and L-B membrane important to lipid metabolism. The effect of 1,25(OH)2D3 on the mRNA for Ca2+-ATPase will be studied. Changes in these parameters as related to enterocyte differentiation will be emphasized. Since there is evidence that 1,25(OH)2D3-dependent calcium transport is, in fact, dependent on enterocyte differentiation, it seems an ideal system for increasing our understanding of differentiation and tissue organization in relationship to a specific intestinal function. Regulation of intestinal calcium transport is fundamental to calcium homeostasis. Therefore, these studies will help in understanding the pathophysiology of osteoporosis, kidney stones, and the effects of intestinal diseases on calcium absorption and calcium metabolism.